Dendritic cell tumor injection (dcti) therapy

ABSTRACT

The invention relates to a method of treating tumor cells within a patient wherein immature dendritic cells developed from the patient&#39;s monocyte cells and a lymphocyte cultured medium (LCM) adjuvant are introduced into the patient directly into the patient&#39;s tumor cells. The immature dendritic cells and LCM adjuvant combine with the antigens in the tumor cells to form a cancer vaccine, thereby immediately treating the tumor cells of the patient. The invention also provides a precursor treatment step of treating the patient with radiation therapy or a chemotherapy regimen.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part application to U.S. patent application Ser. No. 11/227,374, filed Sep. 15, 2005, which claims priority to U.S. Provisional Patent Application 60/610,822 filed Sep. 17, 2004, both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a tumor therapy that includes the injection of immature dendritic cells and adjuvant directly into the patient's (a human or an animal) tumor tissue, which presents antigenicity as a vaccine antigen at the injection sight. Conjugation of these elements within the tumor tissue rapidly induce and activate the patient's immune system to dramatically reduce and/or eliminate tumor cells. Most adjuvants, which augment the immune response, can be directly injected with immature dendritic cells to the tumor tissue to achieve the reduction or elimination of tumor tissues.

2. Description of the Prior Art

Immunological adjuvants are used in combination with vaccines to augment the immune response to the antigen. One way in which immunological adjuvants function is by attracting macrophages to the antigen, so that the macrophages can present the antigen to the regional lymph nodes and initiate an effective antigenic response. Adjuvants may also act as carriers themselves for the antigen, or may influence the immune response by other mechanisms such as depot effect, cytokine induction, complement activation, recruiting of different cell populations of the immunological system, antigen delivery to different antigen presenting cells, regulation of the expression of HLA class I or class II molecules and the stimulation to produce different antibody subtypes. Many of the newer vaccines are only weakly immunogenic and thus require the presence of adjuvants.

Materials having adjuvant activity are well known. Alum (Al(OH)₃), and similar aluminum gels are adjuvants licensed for human use. The adjuvant activity of alum was first discovered in 1926 by Glenny (Chemistry and Industry, Jun. 15, 1926; J. Path. Bacteriol, 34, 267). Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.

One line of research in the development of adjuvants has been directed to the study of dendritic cells. Dendritic cells (DC) are professional antigen presenting cells (APC) that have the unique capacity to initiate primary immune responses in vivo and in vitro. They are derived from myeloid (DC1) or lymphoid (DC2) precursors and are distributed in their immature form throughout the body in tissues that commonly encounter environmental pathogens (skin, mucus membranes, gut epithelia, etc.). Whereas DC1 and DC2 comprise a small percentage of the total number of mononuclear cells in the peripheral circulation, DC1 precursors in the form of CD14+/CD11c+/HLA-DR+ monocytes are relatively abundant, constituting about 10% to 15% of mononuclear blood cells.

Immature DC express a host of surface structures that are involved in antigen acquisition, DC activation/maturation, and antigen presentation. Once DC encounter antigen, they undergo a maturation process characterized by the up-regulation of HLA class I and II molecules as well as co-stimulatory molecules and interact with cognate receptors on T and B lymphocytes, resulting in the generation of antigen specific cellular and humoral immune responses.

DC are considered to be the primary APC in the immune system. The ability to isolate these cells and/or their precursors and to study them in vitro has added considerable dimension to knowledge of their role in innate and acquired immunity. The classic means of generating human DC in vitro is to isolate and enrich CD14+-monocytes from peripheral blood and culture them for various periods of time in GM-CSF and IL-4 followed by final maturation with a number of cytokines, including IL-2, IL-6, IL-7, IL-13, IL-15, TNFα, IL-10, or with various other agents including lipopolysaccharides, PGE2, type 1 interferons, or double-stranded RNA.

Numerous investigators have shown that these in vitro generated monocyte-derived DC are potent antigen presenting cells (APC) capable of initiating primary and recall antigen-specific CD4⁺ and CD8⁺ T cell responses. Recent in vitro studies have generated a rather extensive body of information regarding the biology of DC1 and shed light on the processes whereby antigen specific immune responses are generated in vivo. In the peripheral tissues, immature DC acquire antigenic materials in the context of danger signals initiating a complex cytokine/chemokine milieu that is generated by DC and other cell types in the vicinity.

Soluble mediators produced by DC may act in an autocrine or paracrine fashion. T cells produce additional cytokines and chemokines following interaction with antigen armed DC, as do other immune cells that are activated by the cytokines released. This complex network of interactions may in turn create an environment that promotes the generation of DC from their monocyte precursors.

It is thought that those adjuvants which promote that maturation of dendritic cells, when administered in combination with a vaccine antigen, will result in more antigen presenting cells presenting the vaccine antigen to T lymphocytes and B cells, thus bolstering the immune response to the vaccine antigen. However, isolation of the most effective vaccine antigen has been extremely difficult since antigenicity of APC has always been subject to its evolution with antigenic drift and/or shift, and therefore many of the newer vaccines are only weakly immunogenic even though dendritic cells and adjuvant are present. The most effective vaccine antigen against the live tumor cells should be used with dendritic cells and adjuvant during a course of treatment to promote and to induce a rather strong immunogenicity.

SUMMARY OF THE INVENTION

The present invention solves the above need by providing the most effective antigenic vaccine antigen with dendritic cells and adjuvant to increase the amount and quality of the immune response against tumor cells.

In an aspect of the present invention, there is provided a method of reduction of tumor cells in tumor tissue of a patient, comprising collecting monocyte cells from the patient, culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells, and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant to the patient. The LCM adjuvant comprises at least three, preferably at least six and more preferably at least ten cytokines selected from eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, ILβ, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα and VEGF.

The immature dendritic cells and LCM adjuvant are administered intratumorally, i.e., directly into the site of the tumor.

Optionally, this method provides treating the patient with chemotherapy, radiation or anti T-cell antibodies prior to the administration of the immature dendritic cells and LCM adjuvant.

In another aspect of the present invention, there is provided a method of reduction of tumor cells in tumor tissue comprising treating a tumor of a patient, with a chemotherapy regimen, collecting monocyte cells from the patient, culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant to the patient. The LCM adjuvant comprises at least three, preferably at least six and more preferably at least ten cytokines selected from eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.

Optionally, this method provides treating the patient with radiation prior to the administration of the immature dendritic cells and LCM adjuvant.

In a further aspect of the present invention, there is provided a method of reduction of tumor cells in tumor tissue comprising treating a tumor of a patient with a radiation therapy regimen, collecting monocyte cells from the patient, culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells, and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant into the tumor tissue of the patient. The LCM adjuvant comprises at least three, preferably at least six and more preferably at least ten cytokines selected from eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.

Optionally, this method provides treating the patient with chemotherapy prior to the administration of the immature dendritic cells and LCM adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

A full understanding of the invention can be gained from the following description of the preferred embodiments when read in conjunction with the accompanying drawings in which:

FIG. 1 shows two protocols for treating patients with tumors according to the methods of the present invention;

FIG. 2 shows a computerized tomography (CT) image of a patient with gastric cancer and liver metastasis before and after treatment according to the methods of the present invention;

FIG. 3 shows a CT image of a patient with upper pharyngeal cancer before and after treatment according to the methods of the present invention;

FIG. 4 shows a CT image of a patient with sigmoid cancer and liver metastasis before and after treatment according to the methods of the present invention;

FIG. 5 shows a CT image of a patient with rectal cancer and lung, pelvic and left cervical metastasis before and after treatment according to the methods of the present invention;

FIG. 6 shows a CT image of a patient with right breast cancer and left chest wall and mediastinal metastasis before and after treatment according to the methods of the present invention;

FIG. 7 shows the effect of LCM on surface marker expression, in which monocytes in PBMCs differentiate to a DC-like phenotype following exposure to LCM;

FIG. 8 shows the effect of LCM on surface marker expression, in which immature monocyte-derived DCs differentiate to a mature-phenotype following exposure to LCM;

FIG. 9 shows that LCM augments CpG-induced maturation and IFNα production by CpG treated plasmacytoid DCs (pDCs);

FIG. 10 shows the effect of LCM treatment on T cell responses in vitro;

FIG. 11 shows T cell responses to vaccines are enhanced following treatment with LCM (ELISPOT);

FIG. 12 shows antibody responses to vaccines are enhanced following treatment with LCM (ELISA);

FIG. 13 provides an outline of an elutriation study;

FIG. 14 shows percent viability following incubation with LCM;

FIGS. 15 A, B provides recall responses and shows that LCM augments response to antigens (CMV, n=2); A: Aph082305 and Aph011006; B: Aph082305 and Aph011006;

FIGS. 16 A, B shows tumor cell lysates (n=2); A: Aph062805; B: Aph011006;

FIGS. 17 A, B shows responses of LCM-treated ‘naïve IL7-IL15-treated’ cells (N=2); A: Aph062805; B: Aph01106; and

FIG. 18 shows a proposed culture system for lymphocytes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “leukocyte cultured medium (LCM)” is synonymous and interchangeable with the term “activated leukocyte medium (ALM).”

As used herein, “patients” in clude mammals, which include humans.

As used herein, the term “therapeutically effective amount” refers to that amount of immature dendritic cells and lymphocyte cultured medium (LCM) adjuvant required to bring about a desired effect in a human or other mammal. In all instances, at its most basic level, the desired effect is a reduction of tumor cells in tumor tissue of the patient when compared to the tumor cells in the tumor tissue of the patient prior to employing the methods of the present invention.

The present invention provides treatment tumor tissue using full antigenic elements, which include antigenicity of both known and unknown antigen presenting cells, by locating them within the live tumor tissue in the human body (or alternatively, the body of an animal). This is in contrast to prior art cultured antigens obtained from tumor cell lines or any process added antigen, which have limited antigencity and outdated antigenic data or potency as a vaccine antigen for the patient's tumor cells. In particular, the present invention relates to a therapy that includes the injection of immature dendritic cells and adjuvant directly into the patient's tumor tissue, which presents antigenic elements as the vaccine antigen at the injection sight. The conjugation of these elements within the tumor tissue rapidly induce and activate the patient's immune system to dramatically reduce and/or eliminate tumor cells. Most adjuvants, which augment the immune response, can be directly injected with immature dendritic cells into the tumor tissue to achieve the reduction or elimination of tumor cells. Such adjuvants may include, without limitation, lipid-based, protein-based and polysaccharides-based adjuvants, such as lymphocyte cultured medium, Marignase, Agaricus, OK432, BCG, Lentinan (shiitake), Reishi, Sarunokoshikake, TNF Meshimakobu, Froint's complete or incomplete adjuvant, LPS, fatty acids, phospholipids, cytokines or a virus.

The present invention provides rapid reduction and/or elimination of tumor cells, which can be visually detected by MRI and/or CT and/or Echo scan within two weeks after the injection. The therapy according to a preferred embodiment of the invention includes the following steps: Step 1: Colleting peripheral blood monocyte cells (PBMC) from a patient; Step 2: Culturing these PBMC with GM-CFS and IL-4 to immature dendritic cells; Step 3: Injecting the cultured immature dendritic cells and an adjuvant into the tumor; and Step 4: Evaluating the tumor in two weeks.

In one particular embodiment, the effectiveness (immuno-response) of this method of treatment can be enhanced by pre-treating the tumor cells using known chemotherapy and/or radiation therapy techniques, which diminish the existing immune system, prior to steps 1-4 described above. In addition, the effectiveness (immuno-response) of this method of treatment can also be enhanced by injecting the tumors cells with an anti T-cell monoclonal antibody prior to steps 1-4 described above (either alone or in addition to the chemotherapy and/or radiation therapy described above).

EXAMPLES

The present invention is more particularly described in the following non-limiting examples, which are intended to be illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art.

Example 1 Treatment with Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant

Six patients, four with stomach cancer and two with colon cancer, were used in this clinical investigation to assess the effect of intratumoral administration of immature dendritic cells (imDCs) with a lymphocyte cultured medium adjuvant (LCMadj). All patients were self-referred, had advanced cancers and progressive disease that had not responded to conventional standard therapies.

1. Methods

Four weeks prior to administration of the imDC and LCMadj, leukapheresis was performed on each patient to collect monocyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of imDCs. Four weeks later, a cocktail was prepared containing between about 10⁷ to 10⁸ imDCs and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 to 50 cc of normal saline was injected into the tumor site of each patient. Four weeks after injection of the cocktail, the patients were evaluated by CT image analysis and measurement of serum tumor markers.

2. Results

Of the six patients in this clinical study, three of the tumors of the patients showed stable disease (SD); defined as showing less than a 20% increase in tumor size and less than a 30% reduction in tumor size, with no increase in serum tumor markers. The tumors of the other three patients showed progressive disease (PD); defined as a 20% or greater increase in tumor size, new metastatic lesions and an increase in serum markers.

Example 2 Pretreatment with Chemotherapy Prior to Injection of Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant

Four patients, three with rectal cancer and one with colon cancer, were used in this clinical investigation to assess the effect of chemotherapy prior to intratumoral administration of imDCs with a LCMadj. All patients were self-referred, had advanced cancers and progressive disease that had not responded to conventional standard therapies.

1. Methods

As shown in FIG. 1, Four weeks prior to administration of the imDC and LCMadj, leukapheresis was performed on each patient to collect monocyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of imDCs. Three weeks later, all patients were administered cytoxan intratumorally. One week later, a cocktail was prepared containing between about 10⁷ to 10⁸ imDCs and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 to 50 cc of normal saline was injected into the tumor site of each patient. Four weeks after injection of the cocktail, the patients were evaluated by CT image analysis and measurement of serum tumor markers.

2. Results

Of the four patients in this clinical study, two of the tumors of the patients showed a partial response (PR); defined as a 30% reduction in the size of the injected tumor, decline in serum markers, no increase in tumor size at other metastatic sites or appearance of new metastasis. The tumor from the third patient showed stable disease (SD), as defined above; and the tumor from the fourth patient showed progressive disease (PD), as defined above.

Example 3 Injection of Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant or Pretreatment with Chemotherapy or Radiation Therapy Prior to Injection of Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant

Twenty patients with advanced malignancies of different types were used in this clinical study to assess the effect of intratumoral administration of imDCs with an LCMadj, chemotherapy prior to imDCs and LCMadj administration or radiation therapy prior to imDCs and LCMadj administration. All patients were self-referred, had advanced cancers and progressive disease that had not responded to conventional standard therapies. 1.

Methods

Four weeks prior to administration of the imDC and LCMadj, leukapheresis was performed on each patient to collect monocyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of imDCs. Three weeks later, three patients received radiation therapy and 11 patients were given chemotherapy (see Table 1) by administering the chemotherapeutic agent intratumorally. One week later, a cocktail was prepared containing between about 10⁷ to 10⁸ imDCs and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 to 50 cc of normal saline was injected into the tumor site of each patient. Four weeks after injection of the cocktail, the patients were evaluated by CT image analysis and measurement of serum tumor markers.

2. Results

As shown in Table 1, of the six patients that did not receive any prior treatment before administration of the imDCs and LCMadj cocktail, the tumors of two patients showed a partial response (PR) (see, for example, FIG. 2); the tumors of two other patients showed no change (NC) from their previous condition (see, for example, FIG. 3); and the tumors from two other patients showed progressive disease (PD) (see, for example, FIG. 4). Of the three patients that had radiation therapy prior to administration of the imDCs and LCMadj cocktail, the tumor from one patient showed no change (NC) from its previous status. The other patient dropped out before they could be evaluated. Of the eleven patients that received chemotherapy prior to administration of the imDCs and LCMadj cocktail, the tumors from three of the patients showed a partial response (PR) (see for example FIG. 5); the tumors from six of the patients showed no change (NC) from their previous condition (see, for example, FIG. 6); and the tumors from two patients showed progressive disease (PD). FIGS. 2-7 show CT images of various cancers and their response to the treatment protocol.

TABLE 1 Pre- Eval- Sex ID Diagnosis Stage treatment uation F 030593 Gastric Ca Op Rec No PR Liver Meta M 011077 Epi pharyngeal Ca Op Rec No NC M 040231 Sigmoid Ca Op Liver, Rec No PD Lung & Urinary Bladder Meta M 040265 Gastric Ca Op Peritoneal Rec No NC Meta M 051585 Gastric Ca Op Mult, Rec No PR Lever Meta M 040402 Rt. Lung Ca Op Rt Chest Rec Radiation Drop Wall Meta Out M 040465 Gastric Ca Op Liver Rec Radiation NC Meta M 040865 Malig. Melanoma of Rec Endoxan PR Gingiva Op Cervical LN Meta F 031180 Rectal Ca Op., Lung Rec Endoxan PR Meta Pelvic & Lt. Cervical LN Meta F 040764 Sigmoid Ca Op Mult. Rec Chemo NC Liver Meta (TAI) F 010863 Rec. of Rectal & Caecal Rec None PD Cancer Op., Lt. Cervical LN Meta F 041095 Rectal Ca Op. Lung Meta Rec Radiation NC Pelvic LN Meta F 040924 Breast Ca Op Skin Meta Rec Endoxan PR F 040520 Breast Ca Op Skin Meta Rec Endoxan NC M 031119 Lt. Pylvic Tumor Op. Lt. Rec Endoxan PD Cervical & Axilla LN Meta F 040558 Rec. of Rt. Breast Cancer Rec. CDDP NC Op., Liver Metastasis M 040325 Malig. Mesothelioma IV Endoxan NC M 041266 Rectal Ca Op Liver Rec Endoxan PD Metastasis F 900182 Rt. Breast Ca Op., Lt. Rec CDDP NC Chest Wall & Medistinal LN Meta F 041264 Rec. of Endometrial Rec CDDP NC Cancer op., Pelvic LN Metastasis

3. Discussion

Approximately 80% of the patients showed some degree of tumor regression. Moreover, none of the patients had any adverse reaction to the treatment protocol they were given. In those patients showing tumor regression, this was evident within one month after completion of the treatment protocol and effectiveness of the treatment was observed after over 3 months. The number of cases and percentage effectiveness of the treatment protocols were as follows:

Complete response (CR); defined as a decrease in serum markers to normal levels, complete disappearance of all measurable lesions: 0 (0%)

PR 5 (26%) NC 10 (53%)  PD 4 (21%)

Example 4 Preparation of Lymphocyte Cultured Medium (LCM) for Clinical Application Objective

To develop a clinically acceptable method for the production of LCM from elutriated cell fractions obtained from mononuclear cells (MNC) and generate preliminary data in support of a potential IND submission.

BACKGROUND

A variety of cytokines are known to induce the differentiation and maturation of monocyte-derived dendritic cells (DC). Soluble factors found in cell-free supernatants from monocyte and anti-CD3-activated T cells have been found to increase the expression of activation and maturation markers. In this laboratory, earlier studies showed that activation of ficolled PBMC with anti-CD3/CD28 beads results in a product that could mature APCs and augment T cell responses. The activated lymphocyte medium contained a mix of cytokines and chemokines known to be important for the development and migration of DC including GM-CSF, TNFα, IFNγ, IL8, MCP-1 and MIP1. When cultured in LCM, purified monocytes and monocytes in whole PBMC preparations developed a DC-like phenotype characterized by the loss of CD14 and upregulation of costimulatory molecules. Immature DC exposed to LCM underwent maturation within 48 h marked by an increase in surface expression of CD40, CD80, CD86, CD83 and HLADR. LCM-treated DC stimulated potent allogeneic PBMC responses and boosted antigen-specific T cell responses to antigens. Enhanced T cell and antibody responses were observed when LCM was co-administered with a variety of vaccines in macaques. LCM represents a potential ‘physiologic’ product for the generation of DCs in vitro as well as vaccine adjuvant; providing a cytokine milieu for DC generation and immune activation in vivo. Data using activated PBMCs as well as activation products developed from elutriated lymphocyte fractions are included in this study.

The cytokine composition of LCM is shown in Table 2.

TABLE 2 Quantities of Cytokines or Chemokines (PBMCs) Quantity (pg/ml) GM-CSF 23000 IFNα 0 IFNγ 31000 IL1β 70 IL2 5900 IL3 1000 IL4 280 IL6 2170 IL8 47970 IL10 660 IL12 10 IL15 0 MCP1 110040 M-CSF 8690 MIP1α 127200 MIP1β 157890 PGE2 1540 RANTES 20640 CD40L 1270 SDF1α 0 TGFβ 0 TNFα 6430

FIGS. 7 and 8 show the effect of LCM on surface marker expression. Regarding FIG. 7, monocytes in PBMCs differentiated to a DC-like phenotype following exposure to LCM. Expression of CD14, HLA-DR, CD40, CD80, and CD86 was analyzed at 0, 3 and 5 days following exposure to LCM. Data represent mean±SEM of 11 experiments and ** indicates p<0.005. Regarding FIG. 8, immature monocyte-derived DCs differentiated to a mature-phenotype following exposure to LCM. Elutriated monocytes were cultured with GM-CSF/IL-4 for 3-4 days followed by addition of media alone, LCM or Maturation Cocktail for 48 hours. Monocytes cultured in cRPMI only were used as a negative control. CD11c+DCs were examined for surface expression of CD14, HLA-DR, CD40, CD83, CD80, and CD86 by flow cytometry. Open histograms represent staining of DC with isotype control mAb, and shaded histograms represent staining of DC with specific mAb.

FIG. 9 shows that LCM augments CpG-induced maturation and IFNα production by CpG treated plasmacytoid DCs (pDCs). Human pDCs (91-96% purity assessed by surface expression of CD123) were isolated using positive BDCA-4 immunomagnetic selection (Miltenyi Biotech, Auburn, Calif.). Typically, 1×10⁸ monocytes yielded 3-4×10⁵ pDCs. The pDCs were adjusted to 0.5×10⁶ cells/ml in DMEM (Life Technologies, Rockville, Md.) containing 10% fetal bovine serum (BioWhittaker, Walkersville, Md.) and cultured at 1×10⁵ cells per well in 96 well round bottom plates. Freshly isolated pDCs expressed an immature phenotype (CD83⁻, low MHC and co-stimulatory molecules). pDCs were matured with CpG2006 (20 μg/ml) for 24 to 48 h. LCM was added at a 25% dilution.

FIG. 10 shows the effect of LCM treatment on T cell responses in vitro. PBMCs were cultured for 24 h with or without antigen and/or LCM (25%), washed to remove LCM and plated for: (A) Recall Responses (re-plated on ELISPOT for 24 hours; (B) Primary Responses (culture for 7 days with media containing IL7 and IL15, cells washed, then replated on ELISPOT with antigen for 24 hours). CMV=cytomegalovirus lysate; cancer cell lines: K=gastric cancer, P=pancreatic cancer, N=renal cell carcinoma, col=colon cancer.

Effect of LCM Immunization with Vaccines on T Cell and Antibody Responses-In Vivo.

Total solubilized protein was measured in pooled LCM samples (BioRad protein assay based on the method of Bradford, absorbance at 595 nm). To determine adjuvant activity of LCM in vivo, 0.3 ml LCM (97.5 ng) was mixed with individual vaccines (hepatitis A=HepA; tetanus diphtheria toxoid=TDT; rabies or prostate specific antigen=PSA) and each vaccine/LCM mixture was injected IM in macaques at four separate sites (right and left arms and thighs). Selected cytokine levels are calculated in Table 3.

TABLE 3 Cytokine/Chemokine Concentrations of Pooled LCM Injected into Macaques ng/ml ng/injection site total ng/injection GM-CSF 310 93 372 IL-4 2.5 0.75 3 IL-5 1.5 0.45 1.8 IL-8 4.3 1.29 5.2 IL-10 3.2 0.96 3.8 MCP-1 3.7 1.11 4.4 IL-1α 0.228 0.07 0.274 IL-1β 0.364 0.11 0.437 IL-12p40 0.313 0.09 0.376

Animals were injected with vaccines alone or vaccines plus LCM and cell and serum samples removed for testing according to the following timeline, shown in Table 4.

TABLE 4 Treatment timeline for animals receiving vaccine or vaccine plus LCM Treatment 0 7 14 21 28 35 42 49 56 Days of injection Vaccine HepA + + + alone TDT + + + Rabies + + + PSA + + + Days samples collected for testing Cells (ELISPOT) + + + + + Serum (ELISA: IgG + + + + + antibodies to HLA class I and HLA class II antigens and vaccines) Vaccine + LCM HepA + + + TDT + + + Rabies + + + PSA + + Cells (ELISPOT) + + + + + + + + + Serum (ELISA: IgG + + + + + + + + + antibodies to HLA class I and HLA class II antigens and vaccines) + = procedure done on indicated day

FIG. 11 shows that T cell responses to vaccines were enhanced following treatment with LCM (ELISPOT). FIG. 12 shows that antibody responses to vaccines were enhanced following treatment with LCM (ELISA).

Table 5 shows detection of HLA Ab in Macaque serum using solid phase ELISA.

TABLE 5 Class I Class II Monkey ID Day of serum collection code 0 7 14 21 28 35 42 49 56 0 7 14 21 28 35 42 49 56 Vaccine only CC8A − − − − − − − − − − CG33 − − − − − − − − − − 98021 − − − − − − − − − − 99E030 − − − − − − − − − − 99061 − − − − − − − − − − Vaccine and LCM LCM-98023 − − − − + + + + + − − − − − − − − − LCM-99E145 − − − − − − − − − − − − − − − − − − LCM-99E107 − − + + + + + + + − − − − − − − − − *GTI, Waukesha, WI; + = positive detection, − not detected Summary: Media from Anti-CD3/CD28 Activated PBMCs:

Contain cytokines and chemokines that are known to influence the generation of immune responses; induces maturation and differentiation of monocyte-derived DCs and pDCs; augments primary and recall antigen specific T cell responses in vitro; and augments antibody and T cell responses to vaccines in non-human primates.

Data Generated from ‘Purified’ Elutriated Lymphocyte-Derived LCM

To determine if LCM production could be adapted to a larger scale process potentially better defined and more easily amenable to FDA guidelines than the use of ficolled whole blood PBMCs, a study on apheresed cells with autologous testing was initiated. MNC were fractionated into different cell types from healthy individuals utilizing a programmable semi-closed cell separation device (Elutra, Gambro BCT) that allows the collection of cells based primarily on size. This system offers obvious advantages including the automated removal of platelets and red blood cells, collection of a large number of enriched cell populations for autologous treatment including monocytes for generation of DCs, and lymphocytes for activation of T cells and LCM. Using a program developed for monocyte collection; we were able to collect upstream fractionated products containing predominantly lymphocytes. Designated as Fractions 2 and 3, these cells were cryopreserved for LCM preparation and testing. Cell profiles of each fraction of each donor were generated by flow cytometry. Cells were activated with either anti-CD3 antibody+ionomycin or anti-CD3/CD28 beads. The media was tested for cytokine composition and its capacity to ‘mature’ dendritic cells (DCs) and augment T cell responses.

Because this study involved the injection to humans of activated cell products, prior to any laboratory studies, the acceptability of culture materials was first determined by enquiry with FDA. It was recommended that GMP-produced serum-free media filed in previous IND's be used; and all media ‘components’ (including cytokines) be well-defined.

Data: Characterization of Apheresis Products Pre- and Post-Elutriation.

The cell number in healthy donor leukapheresis products and lymphocyte recoveries is shown in Table 6.

TABLE 6 mean ± SD* Pre-elutriation Total MNC in product (×10⁹) 7.2 ± 3   Total RBC in product (×10¹⁰) 4.3 ± 1.1 Total lymphocytes in product (×10⁹) 5.6 ± 2.2 Total monocytes in product (×10⁹)  1.2 ± 0.38 HCT (%) 2.2 ± 0.5 Total PLT in product (×10¹¹) 2.7 ± 0.9 Percentage of lymphocytes in product 79.3 ± 2.9  Post-elutriation Cell recovery in lymphocyte-rich fraction^(a) Fraction 2: ~63%; Fraction 3: ~42% Lymphocyte purity^(b) 81-86 ± 3%  *n = 9; ^(a)Percentage of cells recovered in lymphocyte-rich fractions 2 and 3 with respect to cell counts in starting material (manual count) ^(b)Percentage of lymphocytes in lymphocyte-rich fraction determined by CD3+ labling

Phenotype of Fraction 2 and 3 Cells

To verify that the majority of cells in fractions 2 and 3 were lymphocytes, fresh and cryopreserved fractionated cells were phenotyped by labeling with fluorochrome-conjugated monoclonal antibodies against leukocyte cell surface markers. Profiles of cryopreserved cells are shown in Table 7 as in practice stored cells will be used to generate the batches of clinical product.

TABLE 7 Phenotype of Thawed elutriated fractions Fraction 2* Fraction 3** AVE SD AVE SD Viability 87 7 88 15 CD45⁺ 97 2 97 3 CD3⁺ 86 3 81 3 CD4⁺ 41 5 48 10 CD8⁺ 29 6 21.8 7 CD4⁺DR⁺ 8 2 10 2 CD4⁺CD25⁺ 5 0.5 6 1 CD25⁺ 7 1 9 3 CD3⁺CD56⁺ 16 6 19 9 CD3⁻CD56⁺ 7 5 8 5 CD56⁺ 23 10 24 16 CD19⁺ 3 1 4 2 FACScan analysis, *n = 87 (storage time 9-547 days); **n = 45 (storage time = 9-399 days) Cytokine Composition of LCM Derived from Fractions 2 and 3

Culture conditions based on historical data in flasks and plates (Table 8) were tested with fractions 2 and 3 to select the ‘best’ conditions for further clinical process development.

TABLE 8 Culture conditions tested (37° C., humidified, 5% CO₂) Table 6: Culture Fraction 2 Fraction 3 incubation time 48 h 72 h 48 h 72 h CD3-CD28 beads X X X X No beads X — X — CD3-CD28 beads + — X — X IL2 No beads + IL2 — X — — Anti-CD3 coating — X — —

LCM supernatants were collected by centrifugation and stored at 4° C. until assayed. Cytokines were assessed within a single assay for direct comparison using flow cytometry-based technology (BioRad, BD Biosciences) (see Table 7).

Comment: Data suggest that anti-CD3/CD28 stimulation provide a ‘manufacturing’ system which is easy to execute and yields fairly consistent cytokine patterns. The use of beads compared to flask/bag surface coating with antibody may be preferred as beads can be systematically measured, their use subject to less operator error, and ‘generally’ similar cytokine patterns are observed.

Tables 9A and 9B show survey assay on cultures in traditional polystyrene plates or flasks.

TABLE 9A Activation of FRACTION 2 cells Cytokines Produced from Cells Stimulated under Different Conditions (27-Bioplex) (pg/ml) No Anti-CD3 CD3-CD28 No CD3-CD28 CD3-CD28 beads + Ab beads beads beads beads + IL2 IL2 coating 48 h stimulation (n = 2) 72 h stimulation (n = 2) Eotaxin 145 ± 22   7 ± 10 138 ± 60  487 ± 260 18 ± 24 147 ± 94  FGF 39 ± 2  0 ± 0 76 ± 43 162 ± 132 0 ± 0 23 ± 32 G-CSF 16.7 ± 3   0 ± 0 27 ± 11 89 ± 51 1 ± 1 26 ± 16 GM-CSF 1124 ± 140  20 ± 23 3551 ± 2115 5944 ± 2657 60 ± 52 1373 ± 961  IFNγ 42512 ± 13867 0 ± 0 50335 ± 56228 42997 ± 24322 227 ± 60  55600 ± 49000 IP10 86034 ± 39358 254 ± 360 170543 ± 400405 13066 ± 2666  694 ± 327 1183 ± 1006 IL1β 24 ± 2  0.6 ± 0.1 21 ± 15 130 ± 81  14 ± 24 34 ± 10 IL1ra 164 ± 109  92 ± 118 232 ± 195 320 ± 143 140 ± 76.  45.5 ± 10   IL2 7944 ± 1549 0 ± 0 6750 ± 4760 24724 ± 9572  10867 ± 1961  5836 ± 3686 IL4 155 ± 39  0 ± 0 292 ± 320 251 ± 123  8.2 ± 11.3 61 ± 47 IL5 236 ± 225 0 ± 0 423 ± 403 493 ± 188 1.5 ± 0.6 183 ± 103 IL6 1646 ± 526  12.9 ± 18.2 2797 ± 3566 2032 ± 670  18 ± 21 226 ± 155 IL7 0.8 ± 0.8 0.0 ± 0   4.20 ± 2.8  5.00 ± 4.8  0 ± 0 0.50 ± 0.7  IL8 2205 ± 1700 283 ± 350 4892 ± 8372 2197 ± 561  815 ± 37  1408 ± 1092 IL9 1590 ± 1601 15 ± 18 1918 ± 2235 2523 ± 1007 0 ± 0 118 ± 167 IL10 7298 ± 2236 0 ± 0 2122 ± 2349 2289 ± 629  0 ± 0 1385 ± 1033 IL12 3 ± 1 0 ± 0 16 ± 14 0 ± 0 0 ± 0 0 ± 0 IL13 874 ± 534 0 ± 0 1479 ± 772  1574 ± 370  3 ± 3 530 ± 160 IL15   3 ± 0.4   2 ± 0.1 3 ± 2 10 ± 21 1.5 ± 3   0 ± 0 IL17 481 ± 70  0 ± 0 1087 ± 1860 386 ± 293 0 ± 0 25 ± 35 MCP1 36 ± 36 26 ± 37 69 ± 74 70 ± 80 0 ± 0 6 ± 8 MIP1α 21945 ± 0   15 ± 15 17781 ± 6324  19153 ± 0   413 ± 98  6146 ± 6832 MIP1β 13684 ± 13363 812 ± 646 18277 ± 7656  48070 ± 49740 7079 ± 2396 17893 ± 7504  PDGFbb 267 ± 41  0 ± 0 189 ± 169 779 ± 586 28 ± 33 122 ± 172 RANTES 15101 ± 182  322 ± 82  34432 ± 29650  7969 ± 11890 781 ± 33  1338 ± 709  TNFα 3457 ± 1540 0 ± 0 7540 ± 8717 7142 ± 2016 0 ± 0 2631 ± 1052 VEGF 136 ± 81  0 ± 0 242 ± 173 582 ± 478 22 ± 0  117 ± 132

TABLE 9B Activation of FRACTION 3 cells Cytokines Produced under Different Culture Conditions (pg/ml) CD3-CD28 CD3-CD28 No CD3-CD28 beads + beads beads beads IL2 48 h stimulation 72 h stimulation (n = 2) (n = 2) Eotaxin 173 ± 21  12 ± 17 225 ± 9  0 ± 0 FGF 38 ± 1  3 ± 4 56 ± 15 0 ± 0 G-CSF   22 ± 0.52 0 ± 0 33 ± 5  0 ± 0 GM-CSF 2279 ± 746  22 ± 1  5577 ± 1278 4 ± 6 IFNγ 223663 ± 20006  14 ± 21 327345 ± 41111  0 ± 0 IP10 21221 ± 5076  884 ± 459 31794 ± 684  1680 ± 1073 IL1β 45 ± 3    1 ± 0.97 67 ± 24   1 ± 0.94 IL1ra 1969 ± 180  1397 ± 172  2715 ± 1086 1545 ± 495  IL2 5518 ± 1387 0 ± 0 2655 ± 1569 0 ± 0 IL4 267 ± 124 0 ± 0 252 ± 50  0 ± 0 IL5 411 ± 322 0 ± 0 542 ± 273 0 ± 0 IL6 1840 ± 394  40 ± 50 2378 ± 75  35 ± 45 IL7 0.9 ± 0.9 0.05 ± 0.07   2 ± 0.08 0 ± 0 IL8 26859 ± 13919 9081 ± 883  36702 ± 0   17269 ± 11218 IL9 3437 ± 2136 43 ± 3  9363 ± 4575 43 ± 25 IL10 8554 ± 973  0 ± 0 10940 ± 2529  0 ± 0 IL12 27 ± 11 0 ± 0 23 ± 5  0 ± 0 IL13 977 ± 574 0 ± 0 1938 ± 697  0 ± 0 IL15   4 ± 0.8   2 ± 0.2   6 ± 0.9   2 ± 0.01 IL17  1928 ± 200.8 0 ± 0 2860 ± 1255 0 ± 0 MCP1  689 ± 94.6 243 ± 87  936 ± 308 222 ± 177 MIP1α 12242 ± 13722 48 ± 65 21945 ± 0   3 ± 4 MIP1β 13922 ± 16298 822 ± 583 15449 ± 14138 501 ± 443 PDGFbb  227 ± 14.6 0 ± 0 581 ± 96  0 ± 0 RANTES 8405 ± 944  111 ± 22  34085 ± 14533  93 ± 2.4 TNFα 7015 ± 770  0 ± 0 18531 ± 6916  0 ± 0 VEGF  176 ± 90.2 0 ± 0 277 ± 40  0 ± 0

Development of LCM Closed ‘Manufacturing’ Process

There appeared to be no large differences in cellular composition between fractions 2 and 3; however, cell recovery was highest in fraction 2. Fraction 2 cells were selected for further analysis and development in a closed system. A 3-day culture period using anti-CD3-CD28 bead stimulation was selected. Closed FEP VueLife® bags (2 PF-0025, American Fluoroseal Corporation, Gaithersburg, Md.) were used (in part based on our previous DC culture IND work) as they: reduce risk of contamination while allowing easy access to cells; are transparent so cells can be easily monitored; are non-reactive, i.e., no plasticizers, leachables or extractables to affect cell culture; are manufactured to meet FDA approval; allow O₂, CO₂, and N₂ gas transfer. FEP is impermeable to water and allows incubation without water loss; and therefore, there is no need to use humidified chambers which often is a source of contamination;

Five different aphereses from different donors were used to make LCM in a bag system. Cells were cultured in serum-free, phenol-red free XVIVO10 (BioWhittaker) media using syringe loading at 1×10⁶ cells/ml in 15 ml media plus CD3-CD28 beads (Dynabeads, Dynal) at 3 beads to 1 cell. Bags were placed atop wire racks to ensure proper gas exchange and even cell distribution then incubated for 3 days at 37° C.

Following culture, cells and LCM from individual units were collected by removing beads with a Dynal magnet followed by centrifugation (10 min at 400×g). Cells were phenotyped (Table 10) and collected supernatants were assayed for cytokines using 27 Bioplex flow-based analyses (Table 11 A, B).

Characterization of Activation Products Produced in Closed System

TABLE 10 Phenotype of elutriated cells following activation* CD3-CD28 Non-activated activated (i.e., no beads but cells in culture) AVE SD AVE SD Viability 80 4 91 6 CD3⁺ 76 7 73 6 CD4⁺ 42 6 36 9 CD8⁺ 39 18 32 9 HLA-DR⁺ 20 16 14 5 CD25⁺ 71 9 1 1 CD19⁺ 7 7 10 4 Cytokines Released from Activated Cells

TABLE 11A Cytokines found in supernatants from lymphocyte cultures in ‘bag’ system* CD3-CD28 bead activation Apheresis unit (pg/ml) N = 5 APH062805 APH082305 APH112905 APH011006 APH112706 Ave ± SD Eotaxin 78 86 132 160 117 115 ± 34  FGF 79 95 117 134 115 108 ± 21  G-CSF 18 21 31 41 31 29 ± 9  GM-CSF 3409 3619 3595 8189 3446 4452 ± 2091 IFNγ 5240 7474 27957 188081 54453 56641 ± 76098 IP10 6158 9415 54621 87794 1234754 278548 ± 535605 IL1β 7 6 9 18 19 12 ± 6  IL1ra 115 117 160 659 396 289 ± 237 IL2 3161 4510 14896 14896 6676 8828 ± 5680 IL4 241 247 351 1108 254 440 ± 376 IL5 425 358 328 1431 247 558 ± 492 IL6 977 955 3207 11942 2852 3986 ± 4567 IL7 5 6 8 7 6 6 ± 1 IL8 973 759 1216 7404 26397  7350 ± 11006 IL9 531 132 2040 6364 1397 2093 ± 2501 IL10 469 302 591 2625 1424 1082 ± 965  IL12 29 46 12 26 20 27 ± 12 IL13 1804 2718 737 2309 1148 1743 ± 813  IL15 3 3 5 7 5 5 ± 2 IL17 102 118 1855 5808 805 1738 ± 2385 MCP1 37 24 44 138 242 97 ± 93 MIP1α 19153 19153 19153 19153 19153 19153 ± 0   MIP1β 23200 23200 23200 23200 9465 20453 ± 6143  PDGFbb 57 65 70 127 165 97 ± 47 RANTES 76360 73239 15223 64659 37225 53341 ± 26299 TNFα 2026 1801 7344 29476 9507 10031 ± 11373 VEGF 266 577 97 133 108 236 ± 202 *72 h incubation

TABLE 11B Cytokines found in supernatants from lymphocyte cultures in ‘bag’ system* No Beads Apheresis unit (pg/ml) N = 5 APH062805 APH082305 APH112905 APH011006 APH112706 Ave ± SD Eotaxin 78 86 132 160 117 115 ± 34  FGF 79 95 117 134 115 108 ± 21  G-CSF 18 21 31 41 31 29 ± 9  GM-CSF 3409 3619 3595 8189 3446 4452 ± 2091 IFNγ 5240 7474 27957 188081 54453 56641 ± 76098 IP10 6158 9415 54621 87794 1234754 278548 ± 535605 IL1β 7 6 9 18 19 12 ± 6  IL1ra 115 117 160 659 396 289 ± 237 IL2 3161 4510 14896 14896 6676 8828 ± 5680 IL4 241 247 351 1108 254 440 ± 376 IL5 425 358 328 1431 247 558 ± 492 IL6 977 955 3207 11942 2852 3986 ± 4567 IL7 5 6 8 7 6 6 ± 1 IL8 973 759 1216 7404 26397  7350 ± 11006 IL9 531 132 2040 6364 1397 2093 ± 2501 IL10 469 302 591 2625 1424 1082 ± 965  IL12 29 46 12 26 20 27 ± 12 IL13 1804 2718 737 2309 1148 1743 ± 813  IL15 3 3 5 7 5 5 ± 2 IL17 102 118 1855 5808 805 1738 ± 2385 MCP1 37 24 44 138 242 97 ± 93 MIP1α 19153 19153 19153 19153 19153 19153 ± 0   MIP1β 23200 23200 23200 23200 9465 20453 ± 6143  PDGFbb 57 65 70 127 165 97 ± 47 RANTES 76360 73239 15223 64659 37225 53341 ± 26299 TNFα 2026 1801 7344 29476 9507 10031 ± 11373 VEGF 266 577 97 133 108 236 ± 202 *72 h incubation

Comment: Particularly IFNγ, IP10, IL6, IL9, IL10, TNFα and the chemoattractants appear to be produced at the highest concentrations following stimulation with some variability between units. Though fraction 2 is relatively pure, variation could be possibly due to cell types (e.g., NK cells) and their proportion in each fraction.

A summary of the function of these cytokines for reference is given in Table 12. Awareness of the cytokine concentrations prior to experiments may be used to calculate actual cytokine amount in dilutions, enable matched comparisons between donors, and establish a dosing level for LCM application.

TABLE 12 Selected Cytokines and Their Activities* Cytokine Producing Cell Function GM-CSF Th cells growth and differentiation of monocytes and DC IFNγ T cells, NK cells antiviral, anti-tumor activity, immunoregulation; activates APCs, promotes Th1 IP10 (IFNγ inducible activated T cells mediates Ca+ mobilization, chemotaxis protein) IL-2 Th1 cells growth, proliferation, activation IL-4 Th2 cells proliferation and differentiation MHC Class II proliferation IL-5 Th2 cells proliferation and differentiation IL-6 monocytes, differentiation into plasma cells macrophages, antibody secretion Th2 cells differentiation IL-8 macrophages, chemotaxis endothelial cells IL-10 Th2 cells cytokine production activation IL-12 macrophages, B differentiation into CTL (with IL-2) cells activation MHC expression proliferation pathogen elimination MIP-1α macrophages, chemotaxis activated NK, CD8+T, CD4+T MIP-1β activated NK, chemotaxis CD8+T, CD4+T RANTES activated NK, chemotaxis (regulated on activation CD8+T, CD4+T normal T cell expressed and secreted) TNFα macrophages, CAM and cytokine expression, cellular mast cells, NK proliferation, differentiation, cells inflammation, cell death *derived from sources in the literature

The Effects of LCM Produced in Closed System on Autologous Monocytes/DCs

To assess their properties LCM, or activated T (AT) cells, were added to autologous DCs (for 2-3 days or overnight, respectively). The autologous setting was first tested as this would be the likely protocol ‘type’ for immunotherapeutic approval. Treated cells were examined for: (a) viability following culture measured by trypan blue exclusion (FIG. 14); (b) changes in surface marker expression (e.g., CD14, CD40, CD80, CD83, CD86) measured by flow cytometry (Tables 13, 14); (c) effects on T cell responses measured in IFNγ ELISPOT following exposure to CMV and tumor lysates before and after IL7+IL15 expansion (FIGS. 15, 16).

Cell surface marker expression on DCs following exposure to autologous LCM is shown in Table 13.

TABLE 13 % expression CD14 CD40 CD80 CD86 CD83 pre-LCM exposure Day 0 DC (thawed, no additional 20 77 74 86 2 cytokines added) post-LCM exposure Day 2→Day 3 DC alone 20→7 55→31 58→22 73→51  4→5 DC + LCM (50%)  9→23 47→54 42→30 54→70 11→16 DC + LCM (25%) 21→22 64→60 63→39 67→70 12→13 DC + LCM (10%) 22→ND 52→ND 49→ND 49→ND 13→ND DC + non-activated (i.e., no bead)  4→40 42→44 35→36 19→51  6→5 medium DC + maturation cocktail (IL1β, 43→56 64→54 56→52 66→63 27→15 IL6, TNFα, PGE2) n = 3 units; ND = not determined

Table 14 shows cell surface marker expression on DCs* following overnight exposure to autologous activated T cells.

TABLE 14 Culture Marker (% expression) Condition Donor Viability CD14 CD40 CD80 CD83 CD86 DCs APH112706 93 38 79 86 1 98 pre- APH082305 98 2 70 80 0 42 coculture DCs + APH112706 57 31 92 92 78 65 non- activated T cells APH082305 84 12 98 94 95 94 DCs + APH112706 63 5 27 12 4 43 activated APH082305 85 0 83 94 77 94 T cells *Gating set on DCs Refer to cytokine profiles for activation of T cells from these units (Table 9). High IFNγ production was measured following stimulation of APH112706. NOTE: Cytokine release assessed following coculture of DC and non-activated T cells or AT cells** showed the following Culture Condition DC DC + non- alone DC + AT cells activated T cells T cells alone Eotaxin 36 63 0 1 FGF 38 123 41 10 G-CSF 0 19 0 0 GM-CSF 608 536 250 0 IFNγ 23 241 16 0 IP10 70 54621 5256 71 IL1β 1 18 3 1 IL1ra 386 16394 730 0 IL2 0 12 1 0 IL4 56 45 31 0 IL5 1 10 1 1 IL6 25 421 152 45 IL7 4 15 0 0 IL8 248 18225 2542 137 IL9 5 58 6 0 IL10 2 11 2 0 IL12 5 4 0 0 IL13 0 329 0 0 IL15 1 2 1 0 IL17 20 82 31 23 MCP1 8 288 44 0 MIP1α 4 39 9 16 MIP1β 69 821 507 261 PDGFbb 29 849 122 34 RANTES 9 1125 319 673 TNFα 16 243 23 14 VEGF 85 87 20 13 **APH082305; sups from other cocultures have been stored and are available to assay

Comment: DCs incubated with LCM (for 2 or 3 days) demonstrate some upregulation in the maturation marker CD83, as well as changes in costimulatory molecule expression. When autologous activated or non-activated T cells are added (overnight) to DCs in another set of wells, as expected, upregulation of costimulatory markers is observed in both cell populations-except with AT cells from donor APH112706, which showed a negative change in costimulatory molecules. Though difficult to make sweeping statements with such low sample sizes, these changes could be attributable to a number of factors including level of stimulation, receptor activation on T cells, cytokines and or viable status. Viability may not be the issue here as non-activated T cell-DC samples demonstrated equal viability with maintained high DC marker expression. The ‘stimulatability’ of T cells from donor APH112706 shows that CD3-CD28-activation can produce high levels of IFNγ (see Table 9a) which is APC activating and our observation could be due to high activation and ‘spent’ status which occurred prior to our measurement point.

Cytokines released from DC-T cell cocultures underscore the importance of activation levels (IFNγ and chemotactic cytokines). With the addition of antigen and expanded observation points, these measures may prove useful to further characterize and screen individual cells for activation status and potential clinical efficacy, particularly if indicative of differences between induction of immunity or tolerance.

Recall and Primary T Cell Responses-ELISPOT

Description

-   -   1. Supernatants and antigen were added to monocytes and DCs         (designated as APC):         -   i. Source of DCs: cryopreserved/cultured from monocytes (3             days, serum-free DC medium (CellGenix, Germany) GM-CSF (800             IU/ml)+IL4 (500 IU/ml) (CellGenix);         -   ii. Source of monocytes: cryopreserved elutriated rotor-off             fraction;         -   iii. Cell supernatants tested: 50%, 25% and 10% of original             strength from CD3-CD28 bead-activated or non-activated             cells;     -   2. Cultures were incubated for 2 days at 37° C. and washed free         or LCM or non-activated supernatants then placed in IFNγ ELISPOT         assay (see schematic below):         -   a. For recall responses:             -   i. cells were counted; autologous lymphocytes                 (fraction 2) were added at 10 lymphocytes: 1 APC (total                 1.5×10⁵ cells/well) then             -   ii. plated on IFNγ antibody-coated ELISPOT plates,                 incubated for 3 days at 37° C. then plates developed and                 enumerated         -   b. For primary responses:             -   i. Washed cells were cultured in IL7+IL15 (5 ng/ml each)                 for 7 days, then washed and plated on coated ELISPOT                 plates and developed as above.

Table 15 is a schematic of the assay.

TABLE 15 Schematic of Assay Culture Conditions Day 0 Day 2 Day 8 Day 9 DCs and monocytes Culture DCs Harvest APCs, Restimulate Harvest alone and wash and add T cells with cells and +GM-IL4 monocytes: cells: antigen or assay in +25% LCM ±LCM RECALL: Add ELISPOT +10% LCM ±tumor Assay antigen to IFNγ +no bead sup (myeloma portion antigen- +tumor lysate (d0 + d8 8226)(3 × 10⁴ cells in naive cells or d8) cell ELISPOT overnight +GM-IL4 + tumor equivalent PRIMARY: lysate (d0 + d8 or d8) lysate per Expand +25% LCM + tumor well or portion of lysate (d0 + d8 or d8) CMV cells in IL7 + +10% LCM + tumor lysate IL15 for 7 lysate (d0 + d8 or d8) (0.01 mg/well) days +no bead sup + tumor lysate (d0 + d8 or d8)

Results

FIGS. 15 A, B provides recall responses and shows that LCM augments response to antigens (CMV, n=2).

FIGS. 16 A, B shows tumor cell lysates (n=2), in which Fig. A is Aph062805 and Fig. B is Aph-11006.

Comment: Cocultures of either DC preparation with LCM and tumor cells show enhanced T cell responses; however, the response is larger in cultures from donor APH011006 compared to donor APH062805. It is interesting to refer to the cytokine table (Table 9) and compare the differences in the degree of the capacity for IFNγ production following activation between the donors. Though different levels in the number of spots in this type of assay are expected, in vivo potential may be predictable by determining a stimulation index for a particular cytokine. Such an index would prove useful for screening potential positive activity; however, to determine if this is a real response, a larger sample evaluation to include appropriate controls will be necessary. Interestingly, the monocyte-antigen cocultures in donor APH011006 also show a larger response than those in the APH062805 donor (FIG. 16B) possibly due to the capacity for detection of IFNγ in this donor or activity of other cytokines such as TNFγ. Higher TNFγ levels were also present in the LCM of this donor which could ‘push’ the monocyte to a DC. Unfortunately, the phenotype of these cells was not determined due to limited amount of material.

These data warrant future study to determine the cell (maturation) status and how the cytokine levels should be manipulated to control and potentially predict function.

Primary Responses: IL7-IL15 T Cell Expansion

FIG. 17 shows responses of LCM-treated ‘naïve IL7-IL15-treated’ cells (i.e., cells first exposed to tumor on day 8) were enhanced compared to cells exposed to antigen on days 0 and 8.

Comment: LCM added to DCs and monocytes enhanced tumor antigen presentation to antigen-naïve T cells cultured in IL7 and IL15 for 7 days prior to antigen stimulation. The higher response levels compared to short recall responses (FIG. 15) could be due to the cytokines that help to maintain viability of T or APCs (cell viability 78-100%). When IL7 and IL15 antigen-treated expanded cultures were restimulated with antigen, that is, pulsed with antigen both on days 0 and 8, there was a response in LCM-treated APCs above non-treated; however, the responses were lower than that of APCs that had been treated with GM-IL4. The LCM data may indicate the presence of suppressive factors or optimal levels of cytokine were present-absent and should be adjusted. This data is reported from two different donors. Expanded studies would be valuable to better characterize the responding cells functionally and phenotypically.

Though it may appear that using a few cytokines would be ‘easiest’ to generate a desired immune response, it may be that the mix of cytokines found in LCM will be the most potent; mimicking a true physiological response and demonstrating that cytokine interactions are essential in optimizing functional activity.

SUMMARY

In this protocol, elutriated fractions 2 or 3 may be used for activation. The greatest number of lymphocytes were collected in fraction 2 (Table 6). There were fairly consistent results between the two fractions (Table 9); however, purity in fraction 3 may be an issue if cell levels in the starting units do not meet optimal elutriation criteria. That is, if the starting total cell number (i.e., ≧5×10⁹ cells) or monocyte count (i.e., ≧1×10⁹) falls below the recommended level for the cell separator, cell fractionation patterns can shift and result in heterogeneous cell distribution in later fractions.

Fractionated or lymphocyte-enriched cell populations permit ‘controlled’ activation as measured by the composition of cell products in the LCM. Cytokines, particularly GM-CSF, IFNγ, IP10, IL2, IL6, IL8, IL9, IL10, IL13, MIP1α, MIP1β, RANTES, TNFα, were most highly induced at fairly even distributions (Table 13); however, more samples should be evaluated for presentation to FDA.

LCM enhanced the expression levels of costimulatory molecules (e.g., CD40, CD80, CD86, and CD83) on DCs, an indication of the maturation process important to antigen presentation (Table 13).

LCM promoted an ‘adjuvant-like’ effect on DC function. DCs treated in vitro with 50-25% of the original LCM solution were able to stimulate responses to CMV and tumor antigens in recall assays (FIG. 15).

LCM may help APC function and expand antigen-specific T cells (FIG. 16); however, optimal levels of cytokine are currently undefined (Note: compare to cells incubated with the ‘standard’ GM-CSF+IL4 formulation).

Based on preliminary results, elutriated cells appear to be a good source for the preparation of LCM in the autologous setting. Note PBMC preparations and elutriated fractions were not directly compared from the same donors in “side-by-side” studies. Stimulated PBMCs, presumably due to the presence of monocytes or possibly platelets, do appear to express some cytokines (e.g., MCP1) not seen at high levels in the elutriated cells which could endow a more robust adjuvant effect.

Further development of the production of LCM or cells is warranted, in which a closed system design illustrated in FIG. 17 could be applied to clinical use.

FIG. 18 shows a proposed culture system for lymphocytes.

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims. 

1. A method of reduction of tumor cells in tumor tissue of a patient comprising the steps of: collecting monocyte cells from the patient; culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells; and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant to the patient, said LCM adjuvant comprising at least three cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 2. The method according to claim 1, wherein said LCM adjuvant comprises at least six cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 3. The method according to claim 2, wherein said LCM adjuvant comprises at least ten cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 4. The method of claim 1, wherein the patient is a human.
 5. The method according to claim 1, wherein said immature dendritic cells and LCM adjuvant is administered intratumorally.
 6. The method of claim 1, further comprising the step of treating the patient with chemotherapy prior to the administration of the immature dendritic cells and LCM adjuvant.
 7. The method of claim 1, further comprising the step of treating the patient with radiation therapy prior to administration of the immature dendritic cells and LCM adjuvant.
 8. The method of claim 1, further comprising the step of treating the patient with anti T-cell monoclonal antibodies prior to the administration of the immature dendritic cells and LCM adjuvant.
 9. A method of reduction of tumor cells in tumor tissue comprising the steps of: treating a tumor of a patient with a chemotherapy regimen; collecting monocyte cells from the patient; culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells; and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant to the patient, said LCM adjuvant comprising at least three cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 10. The method according to claim 9, wherein said LCM adjuvant comprises at least six cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 11. The method according to claim 10, wherein said LCM adjuvant comprises at least ten cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 12. The method according to claim 9, wherein said immature dendritic cells and said leukocyte cultured medium (LCM) adjuvant is administered intratumorally.
 13. The method of claim 9, wherein the patient is a human.
 14. The method of claim 9, further comprising the step of treating the patient with radiation therapy prior to the administration of the immature dendritic cells and LCM adjuvant.
 15. A method of reduction of tumor cells in tumor tissue comprising the steps of: treating a tumor of a patient with a radiation therapy regimen; collecting monocyte cells from the patient; culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells; and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant into the tumor tissue of the patient, said LCM adjuvant comprising at least three cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 16. The method according to claim 15, wherein said LCM adjuvant comprises at least six cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 17. The method according to claim 16, wherein said LCM adjuvant comprises at least ten cytokines selected from the group consisting of eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF.
 18. The method according to claim 16, wherein the immature dendritic cells and the LCM adjuvant is administered intratumorally.
 19. The method of claim 16, wherein the patient is a human.
 20. The method of claim 16, further comprising the step of treating the patient with chemotherapy at a time prior to introducing the immature dendritic cells and LCM adjuvant into the tumor tissue. 